aot2022v55i2

Acta Oncologica Turcica 2022; 55: 77-84
79
01.01.2019 and 01.08.2021 due to HBOC
syndrome were included in the study
according to criteria of the National
Comprehensive Cancer Network (NCCN)
[17]. The criteria used to access the test are:
individual who have a pathogenic/probably
pathogenic variant in their cancer
susceptibility genes in any of their relatives,
breast cancer diagnosed ≤45 years, breast and
ovarian cancer, multiple primary breast
cancers either in one or both breasts, male
breast cancer triple negative (estrogen
receptor-negative, progesterone receptornegative,
and HER2/neu-negative breast
cancer diagnosed <60 years, bilateral breast
cancer with the first diagnosed <50 years,
personal history of breast cancer and one close
blood relative with breast cancer <50 or
ovarian cancer or pancreatic cancer. The mean
age of the patients was 45 years (32-71 years).
This study was conducted in Balikesir
University Medical Faculty Health Sciences
Ethics Committee dated 24.11.2021 and
numbered 2021/260. The study was evaluated
as a research file and it was decided that it was
scientifically and ethically appropriate.
Genetic Analysis
Sequence analysis of the BRCA1 and BRCA2
genes, respectively, was studied from the
patients. Then, BRCA1 and BRCA2 gene
deletion duplication analysis and/or multigene
panel associated with cancer
susceptibility were studied from patients in
whom no mutation was detected. The results
of familial segregation analysis of patients
with mutations were not included in this
study.
DNA extraction
Approximately 2 ml of venous blood was
collected from all patients in an EDTA tube.
DNA was isolated from 200 μl peripheral
blood samples from patients using QIAamp
DNA Blood Mini Kit (Qiagen Inc.).
BRCA1/2 gene next-generation sequencing
All the coding exons, with flanking intron
regions, of the BRCA1/2 gene were amplified
by the polymerase chain reaction and
sequenced using next-generation sequencing
www.actaoncologicaturcica.com
(NGS). After library enrichment (MiSeq
Reagent Kit v2, MS-102-2003) and quality
control, the samples were sequenced using the
MiSeq platform (Illumina, San Diego,
California, United States). Raw reads were
quality trimmed with Trimmomatic and were
mapped to reference human genome (hg19)
with using BWA (Burrows-Wheeler
Alignment Tool). Duplicates were removed
using SAMTools and realignment across
indels and base quality recalibration were
performed with GATK. Annotation of
detected variants were performed using
Illumina BaseSpace Variant Interpreter,
InterVar, Franklin, VarSome, ClinVar,
OMIM, and Pubmed. Variants with a
frequency higher than 0.5% were filtered out.
dbNSFP (contains SIFT, PolyPhen-2, LRT,
Mutation Taster) was used to predict the
pathogenicity about the deleteriousness of
variants. Rare variants were classified
according to the ACMG/AMP variant
interpretation framework.
BRCA1/2 gene Deletion/Duplication Analysis
BRCA1 gene exon deletion/duplication was
investigated by multiplex ligation-dependent
probe amplification (MLPA) analysis.
SALSA® Probemix P002 BRCA1 MLPA®
kit was used to analyze all the coding exons as
described by manufacturer’s recommendation
(MRCHolland, Amsterdam, Netherlands).
The reactions were run and analyzed on an
ABI Prism 3130xl DNA Sequencer (Applied
Biosystems, Foster City, CA, USA) and
Coffalyser.Net data analysis software.
BRCA2 gene exon deletion/duplication was
investigated by multiplex ligation-dependent
probe amplification (MLPA) analysis.
SALSA® Probemix P090 BRCA2 MLPA®
kit was used to analyze all the coding exons as
described by manufacturer’s recommendation
(MRCHolland, Amsterdam, Netherlands).
The reactions were run and analyzed on an
ABI Prism 3130xl DNA Sequencer (Applied
Biosystems, Foster City, CA, USA) and
Coffalyser.Net data analysis software.
Targeted multigene panel analysis
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